Synthesis of biotin and related compounds



Patented Nov. 22, 1949 SYNTHESIS OF BIOTI N AND RELATED COMPOUNDS MosesWolf Goldberg, Upper Montclair, and Leo H. Sternbach, Montclair, N. J.,assignors to Hoffmann-La Roche Inc., Roche Park, Nutley,

N. .l., a corporation of New Jersey No Drawing. Application July 24,1947, Serial No. 763,447

Our invention relates to a new methodior the synthesis of biotin andrelated compounds, aswell as to intermediates therefor. Moreparticularly,

14 Claims (Cl. 260 309) compounds, we employ a thiolactone which can berepresented by the following Formula II:

it is concerned with the productionfof higher homologs of biotin havinga side chain of from I five to seven methylene groups and a terminalCH-CH carboxyl group in 2-position of the biotin nucleus, g J instead offour methylene groups and a terminal (H) carboxyl group as in the caseof biotin itself. We have designated the compound which contains InFormula II the Tadlcals R and in the five methylene groups in theside-chain homobiotin, the compound containing six methylene groupsbis-homo-biotin and that containing seven methylene groupstris-homo-biotin.

In our previous application Serial No. 673,642,, filed May 31, 1946, ofwhich the instant application is a continuation-in-part, we havedisclosed a new method for the synthesis of biotin. We have now. foundthatthis method may be eX-- tended to the production of higher homologsof biotin, such as the homo, bis-homo-, and trishomo-biotin abovereferred to. In application, Serial No. 763,446, filed July 24, 1947, wehave disclosed a method for the synthesis of nor-biotin, which has aside-chain of three methylene groups I and a terminal carboxylgroup in2-position, this being a lower homolog of biotin.

Nor-biotin, and in particular the new higher homologs of biotindisclosed in the present application are biologically importantcompounds. All of them displaya pronounced anti-biotin efiect whentested with Saccharomyces cerevz'siae and Lactobacillus casez',homo-biotin being the most active one. The activity against bothorganisms makes them unique among biotin antagonists, for none of theknown anti-biotins can prevent the growth of Sacchdromyces cerevisiae toany sig nificant extent. In fact, some of them, for example,desthiobiotin and biotin sulfone, while active against Lactobacilluscasei, are growth stimulants for the yeast organism.

In general, our present invention is concerned with the production ofbiotin and its homologs which can be represented by the followingFormula I:

C O HN NH $11-$11 I CH2 CH(CHB)11COOH idazolidone nucleus stand forhydrogen and radicals which can be replaced by hydrogen. Thus, either orboth of the N atoms may carry hydrogen-replaceable radicals, as aralkylradicals, for

instance, u-aralkyl radicals like benzyl and aor ring-substitutedbenzyl, for examplad-methyl, u-ethylbenzyl, o-methyl-benzyl, p-ethyl-benzyl,

. p-methoxy-benzyl, p-ethoxy-benzyl; or acyl radicals as, for example,lower acyl radicals such as :acetyl, propionyl, butyryl, and the like,at least one of the R and R radicals being a hydrogenreplaceableradical.

Methods of producing the thiolactone (II) are already described in ourapplication, Serial No. 673,642 and application, Serial No. 761,444,filed July 16, 1947. Other intermediates employed in the preparation ofthis compound (II) are also described in application, Serial No.744,152, filed April 26, 1947. The instant application is acontinuation-in-part of the above mentioned appli*' cations andapplication, Serial No. 763,446, filed July 24, 1947.

In general, to produce the thiolactone (II), a meso-diamino-succinicacid, such as meso-bisbenzylamino-succinic acid, is reacted in analkaline solution with phosgene to form animidazolidone-(Z)-cis-4,5-dicarboxylic acid. The last mentioned compoundis dehydrated with a dehydrating agent, such as acetic anhydride, toform a 3,4-(2'-keto-imidazolido) -2,5-diketo-tetrahydroiuran. The latteris reduced with zinc in the presence of an aliphatic acid and itsanhydride, such as acetic acid and acetic anhydride, to yield a3,4-(2-keto-imidazolido)-2-keto-5-acyloXy-tetrahydrofuran. By reactingthe last mentioned product with hydrogen sulfide in an acidifiedsolution, for instance, in an organic solvent acidified with hydrogenchloride, and then with a salt of hydrogen sulfide, such as sodium orpotassium hydrosulfide, followed by reduction, as with zinc and aceticacid, the thiolactone (II) (I) S is obtained. This procedure isdescribed in more i detail in the applications already referred towherein y represents an integer from 3 to 7. above.

As a starting material in our synthesis of these The following fiowsheet will serve to illustrate significance as in Formula II above, n inFormulae .alkoxy-alkyl-magnesium halogenide having, for

R and n having the same significance as already 2,489,236 3 4 the stepsinvolved in the synthesis of biotin and 2-(w-a1koXy-a1ky1) thiophane(III). The latter its homologs from the thiolactone (II). is dehydratedby treatin it with a dehydrating CH1! JJH-(CHaMCOOH agent, such asacetic acid, to produce a 2-(40- alkoxy-alkylidene) -thiophane (IV),which is hy- III, IV, and V, represents an integer from 3-7, mdrogenated to form a z-(w-alkoxy-alkyl) -thioin Formulae VI-Ifl and IArepresents an integer phane (V). The latter can then, for example, befrom 4-7, R stands for a lower alkyl radical, such treated to r p n f hhy pl as methyl, ethyl, propyl, and the like, and Hal l r ps by hydro n,if it contains two such stands for halogen, groups. TheZ-(w-alkoxy-alkyl)-thiophanes (V), The thiolactone (II) is reacted Withan wwhich contain at least four methylene groups in the side chain, arethen converted into the corexample, the following formula; 7,0responding compounds selected from the group of hose having in place ofthe alkoxy radical a R2 'O (CH2) MgHa1 halogeno, cyano, carboxyl,CII(COOa1kyl)2 or CH(COOH)2 radical. For this purpose, these 2-(w-alkoxy-alkyl) -thiophanes are first treated In the above scheme, Rand R have the same indicated and Hal standing for chlorine, bromine oriodine. The reaction product is a 2-hydroxy- 216 with hydrogen halide,such as hydrogen bromide orhydrogen chloride, to form the corresponding2-(w-halogeno-alkyl)-thiophane (VI); The last mentioned compound maythen be subjected to two series of reactions. One of these series willbe designated as A and the other as B.

I In series A, which corresponds to the reactions set forth in ourapplication Serial No. 673,642, compound (VI) is treated with a cyanide,such as potassium cyanide, to form a 2-(w-cyano-alkyl) -thiophane (VII).The last-mentioned compound is then saponified, for example, withpotassium hydroxide, to form a 2-(w-carboxy-a1kyl)- thiophane (VIII).The latter is treated so as to replace the hydrogen-replaceable groups Rand/or R with hydrogen to form a compound represented by Formula IA.Where one or both of R and R are benzyl radicals, this can be accomplished, for example, by debenzylation with sodium in liquid ammonia.Where the hydrogenreplaceable group is an acyl radical, such as acetyl,this can be replaced with hydrogen by hydrolysis.

In the other series of reactions (B), compound (VI) is condensed with adialkyl-metallomalonate as, for example, diethyl sodio-malonate ordiethyl magnesium-malonate, to form a 2- (a dicarbalkoxy-alkyl)-thiophane (IX). The last mentioned compound is saponified, for example,with potassium hydroxide, to form a 2-(w,mdi-carboxy-alkyl) -thiophane(X). The latter is decarboxylated by heating whereby it loses onemolecule of carbon dioxide, to form a Z-(w-carboxy-alkyl) -thiophane(XI) In the last step, the 2-(w-carboxy-alkyl) -thiophanes (XI) are thentreated so as to replace the hydrogen-replaceable radicals R and/or R,in the same manner as in the preparation of compound (IA) from compound(VIII). In this way, there are also formed compounds corresponding toFormula IA.

In proceeding according to the B series of reactions, it will be notedthat by reacting compound (VI) with the dialkyl metallo-malonate, oneadditional methylene group is introduced in the side chain over thatintroduced by the Grignard reaction with the thiolactone (II). In caseof the A series of reactions, the number of methylene groups in the sidechain remains the same as introduced by the Grignard reaction.

It is to be understood that the specification and claims embrace alltautomeric forms of the compounds named or shown. Moreover, it will beunderstood that the compounds having a single hydrogen-replaceableradical, represented by R or R, as benzyl or acyl, can obtain thisradical either in the 1' or the 3' position.

The following examples will serve to illustrate the production ofbiotin, wherein the thiolactone (II) is reacted with with4-ethoxy-butyl-magnesium bromide in place of 4-methoxy-butylmagnesiumbromide as set forth in our application Serial No. 673,642. Thesubsequent steps correspond to those in the aforesaid application.

EXAMPLE 1 3,4 (1,3'-dibeneyZ-2-keto-imidazolido)-2-hydrowy-Z-W-ethoxy-butyl) -thiophane or (in its open form) 1,3dibenzyl cis 4 mercaptomethyl-Z-(w-ethoxy'ualeryl) imidazoZidone-2 AGrignard solution, prepared from 8.5 grams 4-ethoxy-butylbromide and anexcess of magnesium (2.4 grams) in 15 cc. of ether and 5 cc. of benzene,is diluted with benzene, decanted from the unreacted magnesium and addeddropwise (in about30 minutes) to a boiling, stirred solu- 6 tion of 13.5grams of 3,4-(1,3-dibenzy1-2'-ketoimidazolido)-2-keto-thiophane(thiolactone) in 160 cc. of benzene. The solution is then kept refluxingfor another 2 hours. The mixture is decomposed with ice and dilutesulfuric acid, the organic layer is separated and concentrated in vacuo.The residue is dissolved in methanol and heated to 50 C. with an excessof aqueous sodium hydroxide solution. Ether and water are added, and the2 layers are separated. The alkaline aqueous solution contains theunreacted thiolactone, which is extracted and recovered afteracidification. The ether layer containing the reaction product is dried,and concentrated in vacuo. The residue is recrystallized from a mixtureof acetone, ether, and petrolether, and forms thick plates melting atEXAMPLE 2 3,4 (1,3 dz'benzyZ-Z-lceto-imidaeolido)-2-(wethomy-butylidene) -thiophane A solution of 20.0 grams of3,4-(1',3-dibenzyl- 2 ketoimidazolido)-2-hydroxy-2-(w-ethoxybutyl)-thiophane in 100 cc. aceticacid is refluxed for 1 hours. The solution is concentrated in vacuo, theresidue is dissolved in ether and the solution is washed with dilutesodium carbonate solution. The ether solution is dried and concentratedin vacuo. The oily residue represents the unsaturatedw-ethoxy-butylidene compound. The yield is quantitative. The product canbe recrystallized from a mixture of ether and petrol-ether and formscrystals melting at 5658 C.

EXAMPLE 3 3,4 (1 ',3 dibeneyZ-Z'-keto-imidazolido)-2-(wethoxybutyl)-thiophane The 3,4- 1 ,3 -dibenzyl-2-keto-imidazolido) 2-(w-ethoxy-butylidene) -thiophane (20 grams),

obtained in the previous example, is dissolved in cc. of methanol andhydrogenated in presence of 2040 grams Raney nickel catalyst at roomtemperature and atmospheric pressure. The calculated amount of hydrogenis taken up in 30 hours. Palladium catalysts, as for example palladiumon charcoal, palladium on barium sulfate and palladium oxide, can alsobe used. The catalyst is filtered off and the solution is concentratedin vacuo. The hydrogenation product is used without further purificationfor the following reaction. It can be recrystallized from petrol-etherand forms thin plates melting at 84-85 C.

EXAMPLE 4 3,4 (N-monobenzyZ-Z'Jeeto-imidaeolido) -2- (wethomybutyl)-thz'ophane A solution containing 7.1 grams of 3,4-(l',3 dibenzyl 2keto-imidazolido)-2(w-ethoxybutyl) -thiophane in 20-30 cc. xylene isadded to 20-30 cc. liquid ammonia cooled in a dry ice bath. To thestirred mixture is added sodium (in form of small pieces) until apersisting blue color develops (about 0.78 gram sodium is used). Thelast excess of sodium is destroyed with ammonium chloride, the ammoniais evaporated, and dilute sulfuric acid and ether are added. The mixtureis stirred for a while and then filtered. The bulk of the reactionproduct remains on the funnel; a smaller part is dissolved in the etherlayer of the filtrate. The latter is separated (if Some product,precipitates, ethyl-acetate is 7 added), dried and concentrated invacuo. The residue is recrystallized from acetone-ether, together withthe material obtained by filtration. The product can also berecrystallized from a mixture of acetone, ether and petrol-ether. Fineneedles melting at 159-1605 C. are obtained.

EXAMPLE 3,4 -(N-mon0benzyl Z'Jceto-imidazolidO) Z-(wbromo-butyl)-thz'ophane A solution of 4.6 grams 3,4-(N-monobenzyl-2'-keto-imidazolido) -2- (w-ethoxy-butyl) -thiophane in cc. 32% hydrogenbromide in acetic acid is heated on the steam bath for 3 hours. Thepurple solution is concentrated in vacuo, the residue dissolved in ethylacetate, and treated with water. Ether is added to the mixture, and theprecipitated crystalline promo-compound filtered off, and washed withwater and ether.

The filtrate is diluted, if necessary, with some ethyl acetate todissolve some newly precipitated material. The organic layer is thenseparated and concentrated in vacuo. The crude material on the funneland the residue from the organic layer are combined and used for thenext step.

The bromo-compound can be purified by recrystallization from dioxane,acetone or methanol. Needles melting at 168-169 C. are obtained.

The 3,4- (N-monobenzyl-2'-keto-imidazolido) 2-(w-bromo-butyl)-thiophanecan be converted into biotin in the same manner as set forth in ourapplication Serial No. 673,642.

The following examples will serve to illustrate the method of preparinghomo-biotin.

EXAlWPLE 6 3,4- (N-monobenzyZ-Z' -keto-imidazolido) -2- (w,w-,dz'carboxy-pentyl) -thiophane 1.7 grams of sodium are dissolved in aboiling mixture of 23.1 grams diethyl malonate and 20 cc. dry dioxane.To the boiling solution is added a mixture of 200 cc. dry dioxane and26.56 grams of 3,4- (N-monobenzyl 2-keto-imidazolido) --2-(w-bromo-butyl) -thiophane.

The reaction mixture isstirred and refluxed for 6 hours, then 75 cc. percent potassium hydroxide, 60 cc. water, and some methanol are added, andthe solution stirred and refluxed for 6 hours. The stirring is thencontinued for another 15 hours at room temperature. Water is added andany alkali insoluble by-products are extracted with ether. The aqueousalkaline solution is acidified with hydrochloric acid, and thecrystalline precipitate is filtered off and washed on the funnel withsome ether. The product is dried and used without further purificationfor the next step. It can be recrystallized from acetone with theaddition of some ether. Fine needles melting at 159-161 C. are obtained.

EXAMPLE '7 d,Z-MonobenzyZ-homo-biotin A mixture of 24.5 grams of3,4-(N-monobenzyl- 2-keto-imidazolido) 2 -(w,wdicarboxy-pentyl)thiophane and 150 cc. of ortho-dichlorobenzene (B. P. 179) is refluxedfor five minutes. The decarboxylation takes place within that time. Thereaction product crystallizes out from the cooling solution, and isfiltered of]? after a few hours and washed with actetone and ether. Themother liquors are concentrated and yield another crop of crystals. Thesubstance is completely pure and melts at 193-194 C. It can be O V onrecrystallized from dioxane. 193-194 C. are obtained.

EXAMPLE 8 d,l-Homo-biotin 2.47 g. sodium are added in one portion to acooled (Dry Ice-acetone) and stirred suspension of 18.7 g.d,l-monobenzyl-homo-biotin in a mixture of about cc. of xylene and about400 cc. of liquid ammonia. The excess of sodium is destroyed withammonium chloride, after a lasting blue coloration (15 minutes) appears.The ammonia is evaporated, and water, ice, ether and then dilutehydrochloric acid are added to the residue. The acid suspension isfiltered, and the precipitate is dissolved in 4 liters of boiling water.The solution is filtered at about 40 C., the precipitate being unreactedmonobenzylhomo-biotin. The solution is then extracted 3-4 times withchloroform, in order to recover the rest of the unreacted startingmaterial. This treatment with chloroform causes only precipitation ofhomo-biotin. The chloroform layers are separated, and the dissolvedstarting material is recovered and can again be debenzylated. Theprecipitated homo-biotin is filtered off, the aqueous solution isconcentrated and yields another portion of homo-biotin. The product ispurified by dissolving in ammonium hydroxide and precipitating withhydrochloric acid. It forms needles melting at 218-220 C., and gives amelting point depression with d,l-biotin (M. P. 232-234 C.).

The following examples will serve to illustrate Needles melting at 1 theproduction of bis and tris homo-biotin.

EXAMPLE 9 A Grignard solution, prepared from '73 g.6-ethoxy-hexyl-loromide and an excess of magnesium (10 grams) in '79 cc.ether and 20 cc. benzene, is diluted with benzene, decanted from the.unreacted magnesium, and added dropwise (within about 30 minutes) to aboiling, stirred solution of 100 grams thiolactone of1,3-dibenzyl-4-merca:ptomethyl-5- carboXy-imidaZolidone-Z[3,4-(l',3-dibenzyl-2- keto-imidazoli-do) -2-keto-thiophane] in 1 literbenzene. The mixture is refluxed for another 3 /2 hours, and is thendecomposed with ice and dilute sulfuric acid. organic layer is separatedand concentrated in vacuo, the residue is dissolved in methanol andheated to 56 C. with an excess of aqueous sodium hydroxide solution.Ether and water are added, and the two layers are separated. Thealkaline aqueous solution contains the unreacted zthiola-ctone, which isrecovered after acidification. The ether layer, containing the reactionproduct, is dried and concentrated in vacuo. The compound is obtained asan oil.

The 6-ethoxy-hexyl-bromide employed to prepare the Grignard reagent asabove described is a new compound and can be prepared in the followingmanner:

66.4 cc. of phosphorus tri-bromide are added within about one hour to329 cc. of G-ethoxyhexanol-i. The temperature of the cooled mixturerises during the addition from 0 to +13". Themixture is left at roomtemperature for 40 hours, and is then decomposed with ice. The heavyorganic layer is separated, washed with alkali and water vuntil neutral,and dried with anhydrous sodium sulfate. The wash-solutions areextracted with ether or benzene, and the dried 9 extracts are combinedwith the main fraction. The ether is evaporated and the reaction productis distilled in vacuo. It boils at 98100 C. at 13 mm.; n =1.4545, (14:1160.

The G-ethoxyl-hexanol-l is also a new compound. It can be prepared inthe :Eollowine, manner:

104 grams sodium are introduced in portions into a stirred, refluxingmixture of 606 g. 1,6- hexane-diol and 1.5 liters toluene. When all thesodium has been introduced and most of it has reacted (3-4 hours) withformation of a heavy white precipitate, 600 cc. (960 grams) ethylbromide are dropped in Within 1 hour. The solution is stirred andrefluxed for 2 hours, and the stirring is then continued at roomtemperature for 15 to 40 hours, until the reaction mixture is completelyneutral. More ethyl bromide can be added to complete the reaction. Theprecipitated sodium bromide is filtered off, and is washed with toluene.The filtrate is then distilled in a vacuum of 11 mm. After distillingoff the solvent, a forerun of 1,6-diethoxy-hexane boiling at 86/12 mm.is collected (76 grams). The following main fraction is6-ethoxy-hexanol-1, distilling at 104- 105 C./ 12 mm. The residueconsists of hexanediol and solidifies after cooling (233 grams). 340grams of 6-ethoxy-hexanol-1 are obtained. It is a colorless liquid withn =1.4318, and d4 =0.890.

EXAMPLE 10 3,4- (1 ,3'-dibeneyZ-2' -keto-imidaeolz'do) 2-(w-cthorcy-hemylidene) -thz'ophane 70 g. of3,4-(l,3-dibenzyl-2'-keto-imidazolido) 2-hydroxy-2-(w-ethoxy-hexyl)thiophane, as obtained in Example 9, are refluxed for 1% hours with 350cc. glacial acetic acid. The solution is then concentrated in Vacuo, theresidue dissolved in ether, and washed with sodium hydroxide solutionand water until neutral. The organic layer is separated, dried withanhydrous sodium sulfate, filtered and concentrated in vacuo. The oilydehydration product is used for the next step without furtherpurification.

EXAMPLE 11 3,4- (1 ',3-dzbenzyZ-2-keto-imidazolido) 2-(w-ethoazy-hezryl) -thiophane The oily dehydration product, as obtainedin Example 10, is dissolved in 100 cc. methanol and hydrogenated underatmospheric pressure at room temperature in the presence of 140 g.methanol-wet (about '70 g. dry weight) Haney nickel catalyst. After 24.hours, 2450 cc. hydrogen are absorbed. The hydrogenation is theninterrupted, the mixture filtered, the Raney nickel washed withmethanol, and the filtrate concentrated in vacuo. Theresidue isdissolved in xylene and used for the next step.

EXAMPLE 12 3,4- (N -monobenzyZ-2' -ket-imzdaeolz'do) 2-(w-ethoacy-heryl) -thi0phane To a stirred mixture of liquid ammonia(about 1 liter) and a solution containing 69 grams of 3,4-(1',3-dibenzyl-2'-keto-imidazolido) 2 (w-ethoxy-hexyD-thiophane, asobtained in Example 11, in 250 cc. xylene, sodium is added in smallportions until the blue color persists (about 7.8 grams sodium areused). The excess of sodium is destroyed with ammonium chloride, theammonia is evaporated, and water, dilute hydrochloric acid and ether areadded. The mixture is stirred for a While and then filtered. The bulk ofthe reaction product remains on the funnel and is washed with water andether; a smaller part is dissolved in the ether layer or the filtrate.The latter solution is separated (if some product precipitates, ethylacetate is added), dried and concentrated in vacuo. Ether and acetoneare added to the oily residue and the precipitated reaction product isfiltered off. Both fractions are combined and recrystallized frommethanol. Needles melting at 152453 C. are obtained.

EXAMPLE 13 3,4-(N monobenzyl 2'-keto-imidaz'olido) -2(wbromo-hexyl)-thz'ophane EXAMPLE 14 3,4 (N-monobenzyl-Z'-keto-imidazolido)-2-(wecyano-hemyl) -thz'ophane) A mixture of 12 g.3,4-(N-monobenzyl-2'-ketoimidazolido) -2- (w-bromo-hexyl) -thiophane,5.9 g. potassium cyanide and 450 cc. methanol is refluxed for 18 hours.The mixture is then acidified with dilute hydrochloric acid andconcentrated until most of the methanol has evaporated. Water is added,the precipitated nitrile is filtered off, washed with water and usedwithout further purification for the next step. The filtrate isextracted with chloroform. The chloroform solution is dried, andconcentrated in vacuo. The residue is combined with the aboveprecipitate and used for the next reaction. Upon recrystallization fromdioxane, fine needles melting at 1'73-174.5 C. are obtained.

EXAMPLE 15 3,4- (N monobenzyl-Z'-keto-imidaeolido)-2-(wcarboxy-hexyD-thiophane or d,Z-m0nobenzylbis-homo-bz'otz'n Thenitrile, as obtained in Example 14, is suspended in a solution of 43potassium hydroxide in 350 cc. methanol and cc. water. The mixture isrefluxed for 18 hours and is then concentrated until most of themethanol has distilled off. Water is added, and the alkaline solution isextracted with chloroform in order to remove all neutral material. Thealkaline solution is then boiled up and acidified with concentratedhydrochloric acid. The precipitated reaction product is filtered offafter 24 hours. It is then recrystallized from o-dichlorobenzene,forming needles melting at 174-176 C. Upon recrystallization fromdioxane, needles melting at 174- 176 C. are obtained.

EXAMPLE 16 d,Z-Bz's-homo-biotin dium is destroyed with ammoniumchloride, after alasting blue coloration (15. minutes) appears. Theammonia is evaporated, and Water, ice, ether and then dilutehydrochloric acid are added to theresidue. The acid suspension isfiltered, and the precipitate is extracted twice with about 3 litersboiling water. The undissolved monobenzyl bis-homo-biotin is filteredoil".

[The filtrate yields, after cooling, a precipitate consisting of amixture of monobenzyl-bis-homobiotin and bis-homo-biotin. This mixtureis extracted again with several liters of boiling water, the undissolvedmaterial and the precipitate ob tained after cooling beingmonobenzyl-bis-homobiotin. If the precipitate still contains somedebenzylated material, the procedure is repeated. The'combined aqueoussolutions are extracted 3-4 times with chloroform, in order to removethe dissolved part of the unreacted starting material. The chloroformlayers are separated, and the extracted starting material is recoveredand can again be debenzylated. The aqueous solutions are concentratedand yield bis-homo-biotin as a crystalline residue. The compound can bepurified by dissolving in ammonium hydroxide and precipitatin the boilinsolution with hydrochloric acid. Thin plates melting at 200.5- 202.5 C.are obtained.

EXAMPLE 17 3,4-(N-monobenzyl-2-'keto-imidazolido) -2-(w,wdz'carbozcy-heptyl) -thz'ophane 10.71 gram of sodium is dissolved ina boiling mixture of 9.66 grams of diethyl malonate and cc. of drydioxane. To the boiling solution is added a mixture of '100 cc. drydioxane and 12.0 grams of 3,4-(N-monobenzyl-2-keto-imidazolido) Z-(w-bromo-hexyl) -thiophane.

The reaction mixture is stirred and refluxed for six hours, then 30 cc.50 per cent potassium hydroxide, about 30 cc. water and 30 cc. methanolare added to it, and the stirring and refluxing is continued for sixhours. The mixture is then stirred 'for another hours at roomtemperature. Water is added and any alkali insoluble by-products areextracted with ether. The aqueous alkaline solution is "acidified withhydrochloric acid, and the crystalline precipitat is filtered ofi andwashed on the funnel with water. The product -is dried and can be usedfor the de-- carboxylation without further purification. Uponrecrystallization from acetone with the addition of ether, fine needlesmelting with decomposition at 154 C. and resolidifying at 155 C. areobtained.

EXAMPLE 18 d,l-Mo'nobe'nzyl-trisshomo-biotin A mixture of about 10 gramsof 3,4-(N-monobenzyl 2 keto-imidazolido)2-'(w,w-dicarboxyheptyl)-thiophane, as obtained in Example 17, and 150cc. ortho-dichlorobenzene is refluxed for five minutes. Thedecarboxylation takes place within that time. The reaction productcrystallizes "out from the cooling solution, and is filtered off after afew hours and washed with acetone and ether. The mother liquors areconcentrated and yield another crop of crystals. The substance iscompletely pure. M. P. 192-194 C. Upon recrystallization from dioxane,needles melting at 19.2-194 C. are obtained.

EXAMPLE 19 d,l-Tris-homo-biotin 1.12 grams of sodium are added in oneportion to a cooled (Dry Ice-acetone) and stirred suspensionof 9.2 gramsof monobenzyl-tris-homo-biotin in a mixture of about 50 cc. xylene andabout 200 cc. liquid ammonia. The excess of sodium is destroyed withammonium chloride, after a lasting blue coloration (15 minutes) appears.The ammonia is evaporated, and water, ice, ether and then dilutehydrochloric acid are added to the residue. The acid suspension isfiltered, and the precipitate consisting of monobenzyl-trishomo-biotinand tris-homo-biotin is refluxed with about '6 liters of Water.

The boiling mixture is filtered, and the undissolvedmonobenzyl-derivative is separated. The aqueous solution is cooled toroom temperature and the crystals formed are filtered off. Thisprecipitate melting around 205-210 C. consists of tris-homo-biotincontaminated with a smali amount of the monobenzyl-derivative. In orderto obtain a pure product the same procedure (refluxing with water andfiltering, etc.) has to be repeated with this precipitate once or twicemore. The cold aqueous solutions are extracted with chloroform, theextract is concentrated and yields pure tris-homo-biotin. The substancecan be further purifiedby dissolving in ammonia and precipitating fromthe boiling solution with dilute hydrochloric acid. The completely puretrishomo-biotin forms flat needles melting at 216 C.

d,l-Homo-biotin can also be prepared from d,l-biotin as illustrated bythe following example.

EXAMPLE 20 0.4 gram of d,l-biotin is dissolved at +5 C. in 5 cc. thionylchloride. The excess of thionyl chloride is evaporated in vacuo,"benzene is added and the solution concentrated in vacuo, in ordertofree the'acid chloride completely of any thionyl chloride left. Theoily residue is then dissolved in benzene, and the :solution added to anicecooled ether solution containing 5 m. moles of diazomethane. Themixture is stirred for 10 minutes and is then concentrated in vacuo. Theyellow residue is dissolved in cc. methanol, the solution refluxed, anda suspension of silver oxide in methanol, prepared from 1.0 gram silvernitrataadded in 5 portions over two hours. After addition of someactivated carbon, the mixture is filtered, and the methanol solutionconcentrated to a small volume. .5 cc. 3 .N potassium hydroxide .are nowadded to the residue and the solution heated for one hour. Then it istreated with activated carbon, filtered and acidified. Thed,l-homo-biotin precipitates in crystalline form, and is filtered ofiafter 24 hours. Fine needles melting at,2l9-220 C. are obtained.

In asimilar way, d-homobiotin can be prepared from .d-biotin, asillustrated by the following example.

EXAMPLE 21 1.0 gram of d-biotin is dissolved at +5 C. in 10 cc. thionylchloride. After a short while a crystalline precipitate is formed.Petrol-ether and benzene are added in order to complete theprecipitation. This crystalline product is filtered off and introducedinto an ice-cooled ether solution containing 16 .m. moles ofdiazomethane. The mixture is stirred for 10 minutes and thenconcentrated in vacuo. The yellow residue is dissolved in 100 cc.methanol, and refluxed. A suspension of silver oxide in methanol,prepared from 2.0 grams silver nitrate, is added in 5 portions over twohours. Some activated carbon is now added, the mixture is filtered, andthe methanol solution concentrated to a small volume. cc. 3 N potassiumhydroxide are added, and the solution heated for one hour, then treatedwith activated carbon, filtered and acidified. The d-homo-biotinprecipitates in crystalline form, and is filtered on after 24 hours.Fine needles melting at 234.5-235 C. are obtained. The optical rotation[061D is +89.5i0,5 (1 per cent solution in 0.1 N sodium hydroxide).

EXAMPLE 22 3,4- (1',3-dibenzyl -2'- Iceto imz'dazolido) -2- hydroary-Z-(w-ethomypropyl) -thiophane A Grignard solution, prepared from 13.6 cc.of 3-ethoxy-propyl-bromide and an excess of magnesium (4.8 grams) in 30cc. of ether and 10 cc. of benzene, is diluted with benzene, decantedfrom the unreacted magnesium and added dropwise (in about 30 minutes) toa boiling, stirred solution of 27 grams of 3,i-(1',3'-dibenzyl-2-keto-imidazolido) 2-keto thiophane (thiolactone) in 350 cc. of benzene.The solution is refiuxed for another 3 hours. The mixture is thendecomposed with ice and dilute sulfuric acid, the organic layer isseparated and concentrated in vacuo. The residue is dissolved inmethanol and heated to 50 C. with an excess of aqueous sodium hydroxidesolution. Ether and water are added, and the two layers are separated.The alkaline aqueous solution contains the unreacted thiolactone, whichis extracted and recovered after acidification. The ether layer,containing the reaction product, is dried and concentrated in vacuo. Theresidue is crystallized from ether, petrol-ether. i

The product is soluble in strong alkali and gives a positive mercaptantest with sodium nitroprusside. It can be recrystallized from a mixtureof acetone, ether and petrol-ether. Prisms melting at 114.5-115.5 C. areobtained.

EXAMPLE 23 3,4- (1,3'-dz'beneyZ-2'-keto-imidaeolido)-2-(wethoaiy-propylidene) -thiophane A solution of 20.0 grams of3,4-(l',3'-dibenzyl- 2'-keto-imidazolido) -2-hydroxy-2-(wethoxypmpyl)-thiophane in 100cc. acetic acid is refluxed for 1 hours.The solution is concentrated in vacuo, the residue is dissolved in etherand the solution is washed with dilute sodium carbonate solution. Theether solution is dried and concentrated in vacuo. The oily residuesolidifies after a few hours. It can be recrystallized from petroletherand forms fine needles melting at 62.5- 63.5 C.

EXAMPLE 24 3,4-(1',3'-dibenzyZ-2-lceto imidaeolido) 2 (wethowypropyl)-thiophane The crude 3,4-(1',3'-dibenzyl-2'-ketoimidazolido) -2-(w-ethoxypropylidene) -thiophane (19 grams), obtained in the precedingexample, is dissolved in 150 cc. of methanol and hydrogenated in thepresence of 4; grams of prehydrogenated palladium oxide at roomtemperature and atmospheric pressure. drogen is taken up in about 30hours. The catalyst is then filtered off, and the solution isconcentrated in vacuo. The product solidifies after some time and isused in the crude form for the next step.

Other palladium catalysts, for example, Pd on charcoal or on bariumsulfate, and also Raney nickel can be used instead of palladium oxide.

The calculated amount of hy.

EXAMPLE 25 3,4-(N-monoben zyl Z'Jceto imidaeolido) -2-(wethomy-propyl)-thiophane To a stirred mixture of liquid ammonia (about 20 cc.), and asolution containing 1.4 grams of crude 3,4-(1,3'-dibenzyl-2'-keto-imidazolido) -2- (w-ethoxy-propyl) -thiophane in10-20 cc. xylene, sodium is added in small portions until the blue colorpersists (about 0.166 gram sodium is used). The last excess of sodium isdestroyed with ammonium chloride, the ammonia is evaporated, and dilutesulfuric acid and ether are added. This mixture is stirred for a Whileand then filtered. The bulk of the reaction product remains on thefunnel; a smaller part is dissolved in the ether layer of the filtrate.The ether solution is separated (if some product precipitates, ethylacetate is added), dried and concentrated in vacuo. The residue isrecrystallized from acetone ether, together with the product obtained byfiltration. Needles melting at 136-137 C. are obtained.

The acetyl derivative of this product can be prepared in the followingway: 1 gram of the monobenzyl-compound and 4 grams of barium carbonateare stirred and. refluxed for 1 hours with 20 cc. acetic anhydride and20 cc. acetyl chloride. The mixture is cooled, filtered, concentrated,and the residue is recrystallized from a mixture of benzene andpetrol-ether. Needles melting at -85.5 C.

When 3,4-(1,3'-dibenzylor N-monobenzyl-2'- keto-imidazolido) -2-(w-ethoxy-propylthiophane is halogenated, it does not form thecorresponding 2-(w-halogeno-propyl)-thiophane, but 3,4-(1,3-dibenzyl orN-monobenzyl-2-ketoimidazolido) -1,2-trimethylene-thiophanium halide, asis described in our application, Serial No. 763,446, filed July 24,1947. These two last mentioned compounds, when subjected to an A-seriesof reactions according to the fiow sheet above, .form nor-biotin, andwhen subjected to a B-series of reactions, form biotin. This isdescribed in more detail in our aforementioned application, and isillustrated by the following examples which correspond to Examples 22,23, 25, 26, 28, 29 and 30 of said application:

EXAMPLE 26 3,4-(1',3-dibenzyZ-2-lceto imidazolido)-1,2-trzmethylenethiophamum bromide A solution of 5 grams of3,4-(1',3dibenzyl-2- keto -imidazolido) -2- (w-ethoxypropyl) -thiophanein 50 cc. of an 18 per cent solution of hydrogen bromide in acetic acidis heated for three hours to 60 C. The solution is concentrated invacuo, and treated with Water and benzene. The mixture is cooled to +5C. and the precipitated crystalline reaction product is filtered oif andWashed with benzene and cold Water. A further amount of the product canbe obtained by concentrating the aqueous part of the mother liquors (thebenzene layer contains only impurities). The product can berecrystallized from water. Thick plates melting at 220-222 C. areobtained.

EXAMPLE 27 3,4- (1 ,3-diben2yZ-2-ketoimidazolido)-1,2-trzmethylene-thiophanium chloride 4.2 grams of crude3,4-(1,3'-dibenzyl-2'-ketoimidazolido) -2- (w-ethoXy-propyl) -thiophaneare dissolvedin 50 cc. of 99 per cent formic acid saturated withhydrogen chloride (about per cent). The solution is heated for two hoursto 5060 C., and is then concentrated in vacuo. The residue is dissolvedin water and extracted with ether to remove impurities. The aqueoussolution is concentrated to dryness, and the residue is recrystallizedfrom aqueous acetone. Prisms melting at 158-159 C. are obtained. Theproduct is very soluble in water.

EXAMPLE 28 3,4- (monobenzyl-Z-keto-imidazoiido)-1,2-trimethylene-thiophanium bromide A solution of 5 grams of3,4-(monobenzyl-2'- keto-imidazolido -2- (w-GthOXYPIOPYl) thiophane in50 cc. of an 18 per cent solution of hydrogen bromide in acetic acid isheated for three hours at 60 C. The solution is concentrated in vacuoand treated with benzene and water. The aqueous layer is separated andconcentrated in vacuo. The residue is recrystallized from alcohol, or amixture of alcohol and dioxane. Prisms melting at 167-1675 C. areobtained.

EXAMPLE 29 3,4- (monobenzyl-Z-Iceto-imidazolido)-1,2-trimethyZene-thiophanium chloride A solution of 4.0 grams of3,4-(monobenzyl-2- keto-imidazolido) -2- (w-ethoxypropyl) -thiophane in100 cc. of 99 per cent formic acid saturated with hydrogen chloride iskept at room temperature for 48 hours and is then heated for 2 hours at50 C. The solution is then concentrated is vacuo, and the residue istreated with water. The aqueous solution is filtered, extracted withethyl acetate, in order to remove all unreacted starting material, andis then concentrated in vacuo. The crystalline residue is recrystallizedfrom a mixture of water, ethanol and ether. Prisms melting at 158-160 C.are obtained. The product softens around 130 C. and resolidifies again.

EXAMPLE 30 3,4-(1,3-dibenzyl-2'-Iceto-im'idazolido) -2-(wcyanopropyl)-thi0phane 4.45 grams of 3,4-(1,3-dibenzyl-2-keto-imidazolido)-1,2-trimethylene-thiophanium bromide, 3.2 grams of potassium cyanide,250 cc. of alcohol and 30 cc. of water are refluxed for eight hours. Theclear solution is concentrated on the steam bath to a volume of about 70cc., concentrated hydrochloric acid being added at the same time inorder to destroy the excess potassium cyanide. About 800 cc. of waterare added, and the precipitated oil is extracted several times withchloroform and ethyl acetate. After drying and concentrating the organicextract in vacuo, an oily residue weighing about 4 grams is obtained.This material is used without further purification for the next step ofthe synthesis.

EXAMPLE. 31

dJ-Dibenzyl-nor-biotin About 4 grams of impure 3,4- (1',3'-dibenzyl-2'-keto-imidazolido) -2- (w-cyanopropyl) -thiophane, 11 grams of potassiumhydroxide, 80 cc. of methanol and 50 cc. of water are refluxed for 17hours. The clear solution is concentrated on the steambath to a volumeof about 25 cc. Water is then added and the neutral by-productsextracted with ethyl acetate and chloroform. The aqueous solution isthen acidified with concentrated hydrochloric acid and extracted withethyl acetate and chloroform. The combined extracts are concentrated invacuo, leaving an oily residue of 3.2 grams. This oil crystallizes froma mixture of acetone, ether and petrol-ether. After several suchrecrystallizations, the product melts at 118- 120 C. Large fiat platesare obtained.

EXAMPLE 32 d,Z-Nor-biotin 3.2 grams of crude d,l-dibenzyl-nor-biotin,dissolved in 30 cc. of warm purified ethylene glycol diethyl ether areadded dropwise with stirring into 200 cc. of liquid ammonia. A very finesuspension of the ammonium salt of d,l-dibenzyl-biotin is formed. Thereaction mixture is stirred and 590 mg. sodium are added rapidly insmall pieces. After stirring for 10 minutes, the blue color disappears.The solution is stirred for 20 minutes, then 1.2 grams ammonium chlorideare added, and the mixture stirred again for 30 minutes. This process isrepeated three times in all. After distilling off the ammonia, water isadded, and sunicient concentrated hydrochloric acid to bring thesolution to pH 1. The product is filtered off and washed with chloroformand ethyl acetate. The aqueous filtrate is extracted with chloroform andconcentrated in vacuo until crystals appear. Both precipitates aredissolved with stirring in about 300 cc. boiling water. The hot solutionis filtered from insoluble material, and then stirred with chloroform.The chloroform is separated, the aqueous layer filtered and concentratedin vacuo, until crystallization begins. After standing at 4 C. for 24hours, the product is filtered off, Washed with water and then withether on the suction funnel. M. P. 230-233" C.

On concentrating the aqueous filtrate and cooling, 50 mg. of less pured,1nor-bi0tin are obtained, melting at 227 C. d,l-Nor-'biotin gives amelting point depression of about 20 C. with d,lbiotin.

We claim:

1. A compound of the following general formula:

I CH H wherein R and R stand for a member of the group consisting ofhydrogen and a hydrogenreplaceable radical selected from the groupconsisting of aralkyl and lower acyl radicals, and X stands for a memberof the group consisting of =C(OH) -w-alkoxy-alkyl =C=CH-w-alkoxy-alkyl=CH-w-alkoXy-alkyl =CH-w-halogeno-alkyl =CH-w-cyano-alkyl:CH-w,w-dicarbalkoxy-alkyl =CH-w,w-dicarboxy-alkyl =CH-w-carboxy-alkyland wherein when X is =CH-w-a1koxy-alkyl and =CH-w-halogeno-alkyl, atleast one of R and R is a hydrogen-replaceable radical, and wherein whenX is =CH-w-carboxy-alkyl and R and R are both hydrogen, the alkylcontains one of the following: 3, 5, 6 and 7 carbon atoms.

12. Homo-biotin. 3. Bis-homo-biotin. 4. Nor-biotin. 5. A process whichcomprises reacting a 3,4-

(1'-R, 3-R') -2-keto-imidazolido] -2-keto-thiophane with anw-alkoxy-alkyl-magnesium halogenide to form a3,4-[(1R,3-R)-2-keto-imidazolidol-Z-hydroxy 2 (w-alkoxy-alkyl)thiophane, splitting out one molecule of water from said compound toyield a 3,4-[(1'-R,3-R')-2- keto-imidazolidol -2- (w-alkoxy-alkylidene)thiophane, hydrogenating said unsaturated compound to a3,4-[(1'-R,3'-R') -2-keto-imidazolido]-2-(walkoxy-alkyl)-thiophane,halogenating the last mentioned compound by treatment with a halogenacid and converting the reaction product to a 3,4- (1'-R, 3-R)-2-keto-imidazolido] -2- (w-substituted-alkyl) -thiophane wherein thew-substituout is selected from the group consisting of a cyano,CH(COOalkyl)2, CH(COOH)2 and COOH radical, R and R standing for a memberof the group consisting of hydrogen and hydrogen-replaceable radicalsselected from the group consisting of aralkyl and lower acyl radicals,at least one of R and R being a hydrogen-replaceable radical, andreplacing any of the said hydrogenreplaceable radicals by hydrogen.

6. In a process for producing 3,4-(2'-ketoimidazolido) -2-(w-carboxyalkyl) -thiophanes, the step which comprises replacing in a3,4-[(1-R,- 3'-R') -2'-keto-in'iidazolidol -2- (w-carboxyalkyl)thiophane, wherein R and R stand for a member of the group consisting ofhydrogen and a hydrogen-replaceable radical selected from the groupconsisting of aralkyl and lower acyl radicals, at least one of R and Rbeing a hydrogenreplaceable radical, any of the said hydrogenreplaceableradicals with hydrogen.

7. In a process for preparing 3,4-(2'-ketoimidazolido) -2-(w-carboxyalkyl) -thiophanes, the steps which comprise reacting a3,4-[(1'-R, 3'-R') -2-ket0-imidazolido] 2 (w-halogeno-alkyD-thiophanewith an alkali metal cyanide to form a, 3,4-[ (1-R,3'-R)-2'-keto-imidazolidol-2- (w-cyano-alkyl)-thiophane and hydrolyzing thelatter compound to form a 3,4[(1-R,3'-R) -2- keto-imidazolido] 2 (wcarboxy alkyl) -thiophane, R and R standing for a member of the groupconsisting of hydrogen and hydrogen-replaceable radicals selected fromthe group consisting of aralkyl and lower acyl radicals, at least one ofR and R being a hydrogen-replaceable radical.

8. In a process for producing3,4-(2'-keto-imidazolido)-2-(w-carboxyalkyl) thiophanes, the steps whichcomprise reacting a 3,4-[(1-R, 3-R') -2-keto-imidazolidol 2(w-halogeno-alkyl)-thiophane with a dialkyl metallo-malonate to form a3,4-[(1-R, 3-R)-2'-keto-imidaz0- lido] -2- (w,w-dicarbalkoxy-alkyl)-thiophane, hydrolyzing and removing one carboxy group from the lastmentioned compound to form a 3,4- [(1-R,3'-R') 2' keto-imidazolidol 2(w-carboxy-alkyl)-thiophane, R and R standing for a member of the groupconsisting of hydrogen and hydrogen-replaceable radicals selected fromthe group consisting of aralkyl and lower acyl radicals, at least one ofR and R being a hydrogenreplaceable radical.

9. A process as in claim 5, wherein R and R stand for benzyl.

10. A process as in claim 7, wherein R and R stand for benzyl.

11. A process as in claim 8, wherein R and R stand for benzyl.

12. The process which comprises decarboxylating 3,4-(N-monobenzyl-2'-keto-imidaz0lido) -2- (w,w-dicarboxy-pentyl) -thi0phaneto form monobenzyl-homo-biotin, and debenzylating the last mentionedcompound to form homo-biotin.

13. The process which comprises hydrolyzing 3,4-(N-monobenzyl 2'keto-imidazolido) -2- (wcyanohexyD-thiophane to form monobenzylbishomo-biotin, and debenzylating the last-mentioned compound.

14. The process which comprises hydrolyzing 3,4-(1,3'-dibenzyl-2-keto-imidazolido) -2- (w-cyanopropyD-thiophane to formdibenzyl-nor-biotin, and debenzylating the last-mentioned compound toform nor-biotin.

The following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Schneider et a1. Mar. 11, 1947 NumberCertificate of Correction Patent No. 2,489,236 November 22, 1949 MOSESWOLF GOLDBERG ET AL.

It is hereby certified that errors appear in the printed specificationof the above numbered patent requirin correction as follows:

Column 3, Formula II, lower right-hand portion thereof, for

column 5, line 58, after reacted strike out with; column 9, line 5, for6-ethoxylread fi-ethaxy column 14, line 32, after 8585.5 0. insert thewords and! period are obtained; line 34, for ethoxy-propy1- readethozy-propyl)-;

and that the said Letters Patent should be read with these correctionstherein that the same may conform to the record of the case in thePatent Oflice.

Signed and sealed this 4th day of April, A. D. 1950.

9 read THOMAS F. MURPHY,

Assistant Ooma'm'om of PM.

